Myeloid cathelicidin 3

Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra- and inter-species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription-PCR-based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH-1, eCATH-2 and eCATH-3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C-terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin-conserved propiece revealed a polymorphic DNA region with several hybridization-positive fragments and suggested the presence of additional genes. A null eCATH-1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH-1-specific mRNA were found in myeloid cells of gene-positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH-2 and eCATH-3 propeptides, but not of eCATH-1-related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate-stimulated neutrophils. These results thus suggest that eCATH-2 and eCATH-3 are functional genes, whereas eCATH-1 is unable to encode a polypeptide.

Cathelicidins were initially characterized as a family of antimicrobial peptides. Now it is clear that they fulfill several immune functions in addition to their antimicrobial activity. In the current work, three cDNA sequences encoding pheasant cathelicidins were cloned from a constructed bone marrow cDNA library of Phasianus colchicus, using a nested-PCR-based cloning strategy. The three deduced mature antimicrobial peptides, Pc-CATH1, 2 and 3 are composed of 26, 32, and 29 amino acid residues, respectively. Unlike the mammalian cathelicidins that are highly divergent even within the same genus, Pc-CATHs are remarkably conserved with chicken fowlicidins with only a few of residues mutated according to the phylogenetic analysis result. Synthetic Pc-CATH1 exerted strong antimicrobial activity against most of bacteria and fungi tested, including the clinically isolated (IS) drug-resistant strains. Most MIC values against Gram-positive bacteria were in the range of 0.09-2.95 μM in the presence of 100mM NaCl. Pc-CATH1 displayed a negligible hemolytic activity against human erythrocytes, lysing 3.6% of erythrocytes at 3.15 μM (10 μg/ml), significantly higher than the corresponding MIC. Pc-CATH1 was stable in the human serum for up to 72 h, revealing its extraordinary serum stability. These specific features of Pc-CATH1 may make its applications much wider given the potency and breadth of the peptide's bacteriocidal capacity and its resistance towards serum and high-salt environments.

LL-37, the only known human cathelicidin which is encoded by the human antimicrobial peptide (CAMP) gene, plays a critical role in protection against bacterial infection. We previously demonstrated that cathelicidin is induced by 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) in human airway epithelial cells with a resultant increase in bactericidal activity. In this study we identify key factors that co-operate with 1,25(OH) 2 D 3 in the regulation of CAMP. Our results show for the first time that PU.1, the myeloid transcription factor (which has also been identified in lung epithelial cells), co-operates with the vitamin D receptor and CCAAT/enhancer binding protein α (CEBPα) to enhance the induction of CAMP in lung epithelial cells. Our findings also indicate that enhancement of 1,25(OH) 2 D 3 regulation of CAMP by histone deacetylase inhibitors involves co-operation between acetylation and chromatin remodeling through Brahma-related gene 1 (BRG1; a component of the SWItch/sucrose nonfermentable [SWI/SNF] complex). BRG1 can be an activator or repressor depending on BRG1-associated factors. Protein arginine methyltransferase 5 (PRMT5), a methlytransferase which interacts with BRG1, represses 1,25(OH) 2 D 3 induced CAMP in part through dimethylation of H4R3. Our findings identify key mediators involved in the regulation of the CAMP gene in lung epithelial cells and suggest new approaches for therapeutic manipulation of gene expression to increase the antibacterial capability of the airway.